This invention relates generally to compositions and methods useful in the treatment of certain infectious diseases. More specifically, the invention relates to compositions which inhibit proteases, such as malaria cysteine protease. Compounds that inhibit these proteases are useful in the prevention and treatment of malaria, schistosomiasis and other infectious diseases.
Proteases are involved in many important biological processes including protein turnover, blood coagulation, complement activation, hormone processing, and cancer cell invasion. Thus, they are frequently chosen as targets for drug design and discovery. The critical role certain proteases play in the life cycle of parasitic organisms also makes them attractive drug design targets for certain infectious diseases.
Schistosomiasis (bilharziasis) is a parasitic disease caused by schistosomes (blood flukes) that generally live in the veins of the gut and liver of a human host. Adult worms can survive up to 20 years. Female adult worms release thousands of eggs each day, which often find their way to tissues such as liver, brain, and lung, where they cause considerable damage by stimulating the body to form inflammation and scar tissue around them. Most eggs pass through the bladder or wall of the gut. Once outside, they hatch and infect water snails. The parasite multiplies inside the snail, giving rise to thousands of cercariae that exit the snail and swim free in search of a host in which to complete their life cycle.
Malaria is another well known infectious disease caused by protozoa or the genus Plasmodium, which are transmitted by bites of infected mosquitoes. Infection with Plasmodium falciparum, the most virulent human malarial pathogen, is estimated to be responsible for over 1 million deaths each year. The most valuable of the heretofore developed classes of antimalarial drugs are the quinoline-containing compounds, such as chloroquine and mefloquine; chloroquine has been especially effective as both a preventative and a curative. A serious problem in the treatment and control of malaria has been the increasing resistance of populations of P. falciparum to these known antimalarial drugs. In addition, reports of multi-drug resistance makes the search for novel therapies especially urgent. Thus, there remains a great need to identify new compounds that have antimalarial capabilities.
During the trophozoite stage, the parasites infect red blood cells (erythrocytes) where they reproduce asexually. At the completion of each asexual cycle, the red blood cells lyse and merozoites are released which invade new red blood cells. This cycle of lysis and re-infection is responsible for the major clinical manifestations of malaria.
Most anti-malarials are blood schizontocides which are active against the parasites during the intra-erythrocytic stage of its life cycle. Sulphones and sulphonamides inhibit the synthesis of dihydrofolic acid, while biguanides and diaminopyrimidines inhibit the synthesis of tetrahydrofolic acid. Although the mechanism of these anti-malarials is known to involve interference with the parasites"" ability to synthesize nucleic acids [Bruce-Chwatt, L. J., Essential Malariology (Wiley, New York (1985)], the mechanism of action of the quinoline-containing compounds has until recently been surprisingly elusive. Recent work provides evidence that the quinoline derivatives work by interfering with the detoxification activity of a heme polymerase [Slater and Cerami, Nature 355, 167 (1992)], although this has recently been called into question [Dorn et al., Nature 374, 269 (1995)].
During the erythrocytic phase, the parasites degrade hemoglobin as a primary source of amino acids. Rosenthal and co-workers have identified a critical cysteine protease involved in the degradation of hemoglobin, the parasites"" primary source of amino acids [Rosenthal, P. J. et al., J. Clin. Invest. 82, 1560 (1988)]. Blocking this enzyme with cysteine protease inhibitors (such as E-64 and Z-Phe-Arg-FMK) in culture arrests further growth and development of the parasites [Rosenthal, P. J. et al., Mol. Biochem. Parasitol. 35, 177 (1989)]. Because humans (and, probably, most other mammals) do not have an analogous hemoglobinase, inhibition of this protease (either alone or in conjunction with established therapies) provides an attractive strategy for the treatment of malaria. Moreover, inhibition of analogous proteases present in other metazoan parasites would similarly provide potentially valuable techniques for treatment of human and animal patients infected with those parasites.
The aforementioned PCT published application WO 94/06280, the entire disclosure of which is hereby incorporated by reference, describes various classes of metazoan protease inhibitors containing specific structural elements which bind to the S2 subsite and at least one of the S1 and S1xe2x80x2 subsites of the metazoan parasite protease. The protease inhibitors described therein generally include at least two homoaromatic or heteroaromatic ring systems, each comprising one to three rings, joined together by suitable linkers. The linkers concretely exemplified therein in every instance comprise at least two nitrogen atoms as a part of the backbone thereof.
It is an object of the present invention to provide compositions and methods for treatment of malaria and other infectious diseases caused by metazoan parasites.
In accordance with the present invention, there are provided compositions and methods for treating a patient infected with a metazoan parasite by inhibiting the enzymatic action of the metazoan parasite protease, wherein there is employed at least one compound of general formula I
Axe2x80x94Xxe2x80x94B 
wherein
A is a substituted or unsubstituted homoaromatic or heteroaromatic ring system comprising one to three rings which binds to at least one of the S2, S1 and S1xe2x80x2 subsites;
B is a substituted or unsubstituted homoaromatic or heteroaromatic ring system comprising one to three rings which binds to at least one of the S1xe2x80x2, S1 and S2 subsites; and
X is xe2x80x94Cxe2x95x90Cxe2x80x94C(xe2x95x90O)xe2x80x94.
These compositions and methods have particular utility in the treatment of schistosomiasis, malaria, and other infectious diseases. The compositions of the present invention are useful, for example, to inhibit the action of trophozoite cysteine protease, thereby preventing degradation of hemoglobin, the primary source of amino acids for the pathogen that causes human malaria. The methods of the present invention comprise administration to a patient infected with a metazoan parasite of at least one metazoan protease inhibitor of general formula I in an amount effective to inhibit the protease of the metazoan parasite, thereby killing the parasite.
Pursuant to the present invention, compositions and methods for inhibiting the enzymatic action of metazoan parasite proteases comprising an effective amount of at least one metazoan protease inhibitor of general formula I are provided. These compositions have utility in the prevention and treatment of schistosomiasis, malaria, and other infectious diseases. In the case of malaria, the compositions of the invention inhibit the trophozoite cysteine protease. In schistosomiasis, the enzyme inhibited by the compositions of the invention is the adult cysteine protease (or xe2x80x9chemoglobinasexe2x80x9d).
The inhibitors of the present invention have particular utility against the metazoan parasites Plasmodium falciparum (which causes malaria), Schistosoma mansoni (which causes schistosomiasis), and Trypanosoma cruzi (which causes Chagas"" disease). In addition, proteases specific to the following metazoan parasites may also be inhibited by compositions in accordance with the present invention: Giardia lamblia, Entoemeba histolytica, Cryptospiridium spp., Leishmania spp., Brugia spp., Wuchereria spp., Onchocerca spp., Strongyloides spp., Coccidia, Haemanchus spp., Ostertagia spp., Trichomonas spp., Dirofilaria spp., Toxocara spp., Naegleria spp., Pneumocystis carinii, Ascaris spp., other Trypanosoma spp., other Schistosome spp., other Plasmodium spp., Babesia spp., Theileria spp., Anisakis and Isospora beli. 
As described in the aforementioned PCT published application WO 94/06280, optimal ring systems and spacer lengths were determined for protease inhibitors which bind to the S2 and S1xe2x80x2 subsites. In addition, compounds comprising aromatic ring systems characteristic of the inhibitors useful for binding to the S2 and S1xe2x80x2 subsites but joined by shorter linkers than would be appropriate for bridging these subsites were also found to be effective in inhibiting metazoan parasite proteases. Modeling studies with these shorter compounds indicated that one of the aromatic ring systems binds to the S2 subsite and the second aromatic ring system of the shorter compounds binds to the S1 or S1xe2x80x2 subsite (but cannot fill all three subsites fully). By introduction of a third aromatic ring system into the shorter inhibitors it was possible to construct compounds that fit fully all three pockets of the target site of the enzyme (the S2, S1 and S1xe2x80x2 subsites).
Pursuant to the present invention, a broad class of metazoan parasite protease inhibitors have been identified as of particular utility in accordance with the present invention having the general formula
Axe2x80x94Xxe2x80x94B 
wherein
A is a substituted or unsubstituted homoaromatic or heteroaromatic ring system comprising one to three rings which binds to at least one of the S2, S1 and S1xe2x80x2 subsites;
B is a substituted or unsubstituted homoaromatic or heteroaromatic ring system comprising one to three rings which binds to at least one of the S1xe2x80x2, S1 and S2 subsites; and
X is xe2x80x94Cxe2x95x90Cxe2x80x94C(xe2x95x90O)xe2x80x94.
Both homoaromatic ring systems and heteroaromatic ring systems bearing a substituent containing a heteroatom (O, N) and/or heterocyclic systems with charged atoms (in particular, quinoline) are contemplated as within the scope of the invention. In a preferred class of inhibitors in accordance with the present invention, B binds to the S1 subsite. In a preferred subclass of inhibitors, B has the structure xe2x80x94Bxe2x80x2xe2x80x94Xxe2x80x2xe2x80x94Bxe2x80x3, wherein Bxe2x80x2 is an aromatic ring system which binds to the S1 subsite, Bxe2x80x3 is an aromatic ring system which binds to the S1xe2x80x2 subsite, and Xxe2x80x2 is a direct bond or a linker with a backbone of one to three atoms in length.
Each of the ring systems A and B is preferably a one- or two-ring homoaromatic (e.g., phenyl, 1-naphthyl, 2-naphthyl, etc.) or heteroaromatic group which has an affinity for the S2 or S1xe2x80x2 and S1 subsites, respectively. The ring system A or B may be unsubstituted; preferably, however, the ring system bears at least one non-interfering substituent (as hereinafter defined) which does not interfere with, and may actually promote, binding via interactions with the side chains with hydrophobic features, side chains and polypeptide backbone elements with donor and acceptor sites for hydrogen bonds and side chains with formal charges for electrostatic interactions characteristic of the subsite to which it binds.
Particular examples of suitable ring systems include, but are not limited to, the following: phenyl; pyridyl; naphthyl; isoquinolyl; phthalazinyl; coumarinyl; phenanthryl; and quinolyl. Again, all of these ring systems may be unsubstituted or substituted by one or more non-interfering substituents as hereinafter defined. Preferred embodiments of A are phenyl, naphthyl, pyridyl and isoquinolyl; preferred embodiments of B are phenyl and pyridyl.
The choice of a particular structure for use as ring system B depends to some extent on whether the inhibitor is being designed for use to block the S1xe2x80x2 and/or the S1 subsite. In general, it has been determined that for binding to the S1xe2x80x2 subsite, a two-ring system (for example, as described for use as Group A) is more effective than a one-ring system. Multiple-ring systems (and in particular, two-ring systems) as previously described as useful in binding to the S2 and S1 subsites are also presently preferred for binding to the S1xe2x80x2 subsite.
As previously noted, both the ring systems A and B and the linker X may suitably bear one or more non-interfering substituents. For purposes of the present invention, a non-interfering substituent is defined as one which does not interfere with bonding of the ring structures to the active site of the enzyme due to steric and/or electronic factors; in some cases, the presence of particular non-interfering substituents is believed to promote bonding by interaction of these substituents with structural elements of the enzyme in the proximity of the active site. In most instances, the primary consideration with respect to possible substituents is a steric one; for the most part, relatively bulky substituents are not particularly preferred for use in the inhibitors of the present invention. Suitable non-interfering substituents include, but are not limited to, the following: hydroxyl, including protected hydroxyl (i.e., a hydroxyl group which is protected by a suitable art-recognized protective group); lower alkyl; lower alkoxy; amino, mono- and di-(lower-alkyl)-amino; amino-, mono- and di-(lower-alkyl)-amino-lower-alkoxy; xe2x80x94COOH and xe2x80x94COORxe2x80x2, wherein Rxe2x80x2 is lower alkyl or aryl; xe2x80x94NO2; halogen (in particular, Cl, F and Br); aryl (in particular, phenyl and benzyl); and aryloxy (in particular, phenoxy and benzyloxy). For purposes of the present invention, by lower alkyl is meant an alkyl group of one to five, and preferably one to three, carbon atoms.
Pursuant to one particular embodiment of the present invention, the inhibitors have the general formula II
Axe2x80x94Xxe2x80x94Bxe2x80x2xe2x80x94Xxe2x80x2xe2x80x94Bxe2x80x3
and are designed to bind fully to the S2, S1 and S1xe2x80x2 subsites. In these structures, Xxe2x80x2 is exemplified as follows: a direct ring-to-ring bond; a single-atom backbone linker (e.g., xe2x80x94CH2xe2x80x94); and a two-atom backbone linker (e.g., xe2x80x94CH2xe2x80x94Oxe2x80x94 and xe2x80x94Nxe2x95x90Nxe2x80x94). Other Xxe2x80x2 linkers (e.g., those specified as suitable for use as X in the compounds of PCT published application WO 94/06280) would be readily apparent substitutes for these exemplary structures. It is presently preferred that A and Bxe2x80x2 are both phenyl, Bxe2x80x3 is 1-imidazolyl and Xxe2x80x2 is a direct bond. As indicated with respect to the previous structures, however, these examples should be viewed as merely illustrative of the range of structures which may be employed as ring system A, ring system Bxe2x80x2 and ring system Bxe2x80x3, and once again analogous ring systems and substitution patterns other than those depicted herein are clearly contemplated as within the scope of the present invention.
Particular inhibitors suitable for use in the compositions and methods of the present invention include a number of general classes of compounds which have been investigated in some detail. One such class of compounds has the general formula III 
wherein R1 is selected from the group consisting of hydrogen, CF3, hydroxyl, lower alkyl, lower alkoxy and halogen; R2 is selected from the group consisting of hydrogen, CF3, hydroxyl, lower alkyl, lower alkoxy and halogen; R3 is selected from the group consisting of hydrogen, CF3, hydroxyl, lower alkyl, lower alkoxy, nitro, lower-alkylamino, di-lower-alkylamino and halogen; R4 is selected from the group consisting of hydrogen, CF3 and halogen; R5 is selected from the group consisting of hydrogen, CF3, lower alkyl, lower alkoxy and halogen; R6 is selected from the group consisting of hydrogen, CF3, hydroxyl, lower alkyl, lower alkoxy, lower-alkylamino-lower-alkoxy, di-lower-alkylamino-lower-alkoxy and halogen; R7 is selected from the group consisting of hydrogen, CF3, hydroxyl, lower alkyl, lower alkoxy, lower-alkylamino-lower-alkoxy, di-lower-alkylamino-lower-alkoxy and halogen; R8 is selected from the group consisting of hydrogen, CF3, hydroxyl, lower alkyl, lower alkoxy, lower-alkylamino-lower-alkoxy, di-lower-alkylamino-lower-alkoxy and halogen; R9 is selected from the group consisting of hydrogen, CF3, lower-alkylamino-lower-alkoxy, di-lower-alkylamino-lower-alkoxy and halogen; and R10 is selected from the group consisting of hydrogen, CF3, lower-alkylamino-lower-alkoxy and di-lower-alkylamino-lower-alkoxy Preferred compounds of general formula III are those reported in Table I as having an IC50 of less than about 5 xcexcM, more preferably less than about 1 xcexcM.
Exemplary embodiments of the compounds of general formula III are described and the IC50 values obtained therefor in W2 cells infected with a Plasmodium falciparum clone from CDC/Indochina III as hereinafter described are reported in Table I.
In similar tests, compound 44 (RL3119) 
had an IC50 of 3.1 xcexcM (compared to an IC50 of 0.2-0.4 xcexcM for chloroquine).
Another preferred class of compounds having particular utility as inhibitors of metazoan parasite proteases have the general formula IV 
wherein each of R1, R2 and R3 is independently selected from the group consisting of H, lower alkyl, lower alkoxy, nitro and halogen and R is selected from the group consisting of 2- and 3-pyridyl. Exemplary compounds of general formula IV are identified and IC50 values therefor reported in Table II; also included therein is control compound cinnamoylimidazole 48 (RLc00), a commercially-available serine protease inhibitor, which has substantially no activity.
Yet another class of compounds having particular utility as inhibitors for use in accordance with the present invention have the general formula V 
wherein each of R1-R5 is independently selected from the group consisting of H, halogen, lower alkyl, lower alkoxy, lower-alkylamino and di-lower-alkylamino. Exemplary compounds of general formula V are identified and IC50 values therefor reported in Table III.
In similar tests, compound 56 (RL3111) 
had an IC50 of 20 xcexcM (compared to an IC50 of 0.2-0.4 xcexcM for chloroquine).
Yet another class of compounds having particular utility as inhibitors for use in accordance with the present invention have the general formula VI 
wherein X is N or CR6 in which R6 is H or lower alkyl, each of R1-R3 is independently selected from the group consisting of H, halogen, lower alkyl, lower alkoxy, lower-alkylamino and di-lower-alkylamino, and each of R4 and R5 is independently selected from the group consisting of hydroxyl and lower-alkoxy. Exemplary compounds of general formula V are identified and IC50 values therefor reported in Table IV.
Of the compounds tested, 11 compounds had IC50 values at submicromolar levels. The best compound tested, compound 1 (RL388), had an IC50 value of 0.38 xcexcM.
Some trends with respect to the structure-activity relationship in the compounds of the present invention were noted. Substitution with halogen (e.g., F or Cl) on the A ring and with lower-alkoxy (e.g., methoxy) on the B ring generally increased activity; this is illustrated by compounds 1-8, with IC50 values at submicromolar levels. The best embodiments tested thus far include compounds in which R1 and R2 are Cl (the corresponding F-substituted compound 15 had an IC50 of 2.8 xcexcM) and R6 and R8 (compound 1) or R7 and R8 (compound 2) are lower-alkoxy (e.g., methoxy). There were, however, a number of exceptions (see, e.g., compounds 23, 27, 28, 30, 35, 37, 38, 42 and 43).
Some of the compounds tested had limited solubility, even in dimethylsulfoxide; this would be expected to reduce the activity observed. To compensate therefor, it is presently preferred in some embodiments that ring B comprise one or more lower-alkylamino-lower-alkoxy or di-lower-alkylamino-lower-alkoxy (e.g., dimethylaminoethyloxy) groups. Salts of such compounds would typically have increased water solubility.
Electron-withdrawing groups on the A ring and electron-donating groups on the B ring appeared to increase overall antimalarial activity. Again, some exceptions were noted; for example, compounds 9, 11 and 12 (which have electron-donating groups on the A ring) had IC50 values of 1 to 2 xcexcM, although at pH 5 or below the dimethylamino group of compound 11 would likely be positively-charged and therefore act as a strong electron-withdrawing group. In addition, compound 24 with a strong electron-withdrawing nitro group at R3 had poor activity. Compounds wherein the A ring is perhalogenated (e.g., A is pentafluoro- or pentachlorophenyl) or either or both rings comprise at least one CF3 group are of particular interest.
Compounds with hydroxyl groups on both rings (e.g., compound 21) have activity similar to that of hydrazide derivative IV44A disclosed in the aforementioned PCT published application WO 94/06280. They are expected, however, to be more water soluble.
In general, the three-fingered compounds 49-56 with imidazole as the third ring have similar patterns of preference for electron-withdrawing and electron-donating groups as described above for the two-fingered compounds.
Many of the inhibitors employed in accordance with the present invention are either known compounds (some of which are commercially available) or may be readily prepared in a manner known per se from heretofore known and/or commercially-available compounds. The following general schemes illustrate some particularly advantageous synthetic routes; alternative syntheses will of course be readily apparent to those skilled in the field of synthetic organic chemistry.
The compounds of general formula I are known per se and/or may be prepared in an essentially routine manner from heretofore known compounds. While those working in the field would immediately appreciate that other routes for preparation thereof are available, one set of particularly advantageous synthetic schemes for the compounds of general formulas III-VI are as depicted below. 
The inhibitors employed in the compositions and methods of the present invention are typically administered in conjunction with a suitable carrier or adjuvant. It is presently preferred that the inhibitors be administered in an aqueous solution (e.g., buffered saline); however, other suitable excipients and adjuvants would be readily apparent to those of skill in the art. The compositions of the invention may be administered by a wide variety of known routes of administration (e.g., orally, intravenously, subcutaneously, etc.). The inhibitors are suitably administered at a dosage of about 0.01 to about 10 xcexcM, and preferably about 0.01 to about 1 xcexcM, per kilogram of body weight of the patient per day. Of course, as would be appreciated by those of skill in the art, the optimum dosage for treatment of any given parasitic infection with a composition of the present invention comprising one or more specific inhibitors as described herein may readily be determined empirically.
The inhibitors determined to be effective in accordance with the present invention exhibit a surprising specificity for the malarial protease and other evolutionarily-related metazoan parasite proteases. These metazoan parasite proteases are distinct from proteases found in the parasitic hosts (i.e., mammals), particularly with respect to the chemical environments of the active sites of the respective enzymes. In view of the significant differences between corresponding subsites in the mammalian and the metazoan parasite proteases, the inhibitors of the present invention do not in general inhibit the activity of the host""s essential proteases (e.g., cathepsin B).
For example, the malarial enzyme has an asparagine at position 133, a key residue for determining the specificity of bonding at the S2 subsite; for most other non-parasitic cysteine proteases, however, this residue tends to be either branched hydrophobic or alanine. Specific interactions of the inhibitors in accordance with the present invention with the asparagine increase both specificity and potency. Another modulating residue is glutamic acid at position 205 in the malarial enzyme. The side chain rotamer located at the base of the S2 binding pocket is postulated to change depending upon the nature of the interaction at S2. If the substituent is hydrophobic, the glutamic acid points away from the S2 pocket and presumably interacts with solvent; however, when the substituent is basic, the glutamic acid is thought to point towards the S2 pocket and provide a crucial interaction with the positive charge. Inhibitors that exploit this interaction (i.e., those wherein ring system A has some basic characteristics as a part of the ring system and/or by virtue of the substitution pattern) are thus of particular interest.
The invention may be better understood with reference to the accompanying example, which is intended for purposes of illustration only and should not be construed as in any sense limiting the scope of the present invention as defined in the claims appended hereto.